evolution of mammalian sensorimotor cortex: …...evolution of mammalian sensorimotor cortex:...

ORIGINAL RESEARCH ARTICLEpublished: 07 January 2015

doi: 10.3389/fnana.2014.00163

Evolution of mammalian sensorimotor cortex: thalamicprojections to parietal cortical areas in MonodelphisdomesticaJames C. Dooley1, João G. Franca2, Adele M. H. Seelke1,3, Dylan F. Cooke1,3 and Leah A. Krubitzer1,3*

1 Center for Neuroscience, University of California, Davis, Davis, CA, USA2 Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil3 Department of Psychology, University of California, Davis, Davis, CA, USA

Edited by:

Jose L. Lanciego, University ofNavarra, Spain

Reviewed by:

Francisco Clasca, AutonomaUniversity, SpainMarcello Rosa, Monash University,Australia

*Correspondence:

Leah A. Krubitzer, Center forNeuroscience, University ofCalifornia, Davis 1544 Newton Ct.,Davis, CA 95616, USAe-mail: [email protected]

The current experiments build upon previous studies designed to reveal the network ofparietal cortical areas present in the common mammalian ancestor. Understanding thisancestral network is essential for highlighting the basic somatosensory circuitry presentin all mammals, and how this basic plan was modified to generate species specificbehaviors. Our animal model, the short-tailed opossum (Monodelphis domestica), is aSouth American marsupial that has been proposed to have a similar ecological niche andmorphology to the earliest common mammalian ancestor. In this investigation, we injectedretrograde neuroanatomical tracers into the face and body representations of primarysomatosensory cortex (S1), the rostral and caudal somatosensory fields (SR and SC),as well as a multimodal region (MM). Projections from different architectonically definedthalamic nuclei were then quantified. Our results provide further evidence to supportthe hypothesized basic mammalian plan of thalamic projections to S1, with the lateraland medial ventral posterior thalamic nuclei (VPl and VPm) projecting to S1 body and S1face, respectively. Additional strong projections are from the medial division of posteriornucleus (Pom). SR receives projections from several midline nuclei, including the medialdorsal, ventral medial nucleus, and Pom. SC and MM show similar patterns of connectivity,with projections from the ventral anterior and ventral lateral nuclei, VPm and VPl, and theentire posterior nucleus (medial and lateral). Notably, MM is distinguished from SC byrelatively dense projections from the dorsal division of the lateral geniculate nucleus andpulvinar. We discuss the finding that S1 of the short-tailed opossum has a similar patternof projections as other marsupials and mammals, but also some distinct projections notpresent in other mammals. Further we provide additional support for a primitive posteriorparietal cortex which receives input from multiple modalities.

Keywords: marsupial, cortical evolution, multimodal cortex, 3a, S1, posterior parietal, thalamocortical projections,

comparative neuroanatomy

INTRODUCTIONThe emergence of a six-layered neocortex and its diversificationfrom 10 to 12 areas to hundreds of cortical areas is the hallmarkof mammalian evolution (Krubitzer, 2007; Kaas, 2011). Our lab-oratory is interested in how the shared features of all mammalianbrains inform the likely characteristics of the ancestral mam-malian brain, how phenotypic transformations such as additionalcortical areas arise, and the constraints imposed on evolving anddeveloping brains (Krubitzer and Dooley, 2013). There are anumber of cortical areas that are present in all mammalian speciesinvestigated, including primary sensory areas, second sensoryareas, and a few additional cortical areas not exclusively relatedto unimodal sensory processing (for review, see Kaas, 2011).

Interestingly, the organization and number of motor areas inthe cortex is quite different in different mammals, and whetherthe common ancestor of all mammals actually possessed even

a primary motor area (M1) is unclear (Kaas, 2011). While aseparate motor area, rostral to the primary somatosensory area(S1) and containing a complete and mirrored body representa-tion is a feature shared among eutherian mammals, this grouprepresents just one of three major clades of mammals. Further,even in placental mammals the relationship between M1 and S1is variable (e.g., Donoghue et al., 1979; Donoghue and Wise,1982). While only three extant species of Monotremes are avail-able for study, the diversity and relative availability of differentmarsupial species has revealed motor representations that are sur-prisingly divergent from those seen in placental mammals (Karlenand Krubitzer, 2007). Among marsupials, American opossums(order Didelphimorphia) are both the largest order (with over100 species) and form the earliest radiation of marsupials (Kemp,2005), which occurred approximately 150 million years ago(Nilsson et al., 2010). Within this order is a small South American

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Dooley et al. Thalamocortical projections to parietal cortex

marsupial, the short-tailed opossum (Monodelphis domestica).Short-tailed opossums appear to share many characteristics withthe ancestral mammal, including skull morphology, body size,brain size and the ecological niche that they occupy (Rowe et al.,2011), suggesting that these animals could serve as good modelsfor the brain organization of our earliest ancestors.

It is not surprising then, that among marsupials studied,opossums are the only order which have been consistently andconvincingly demonstrated to lack a separate motor cortex inwhich microstimulation evokes movements (Magalhaes-Castroand Saraiva, 1971; Beck et al., 1996; Frost et al., 2000); forreview see (Karlen and Krubitzer, 2007). In all opossum species inwhich microstimulation techniques have been used, movementscould only be initiated by stimulating S1. Specifically, in boththe big-eared opossum (Didelphis aurita) and the Virginia opos-sum (Didelphis virginiana), movements could be elicited whenstimulating separate locations throughout the entire extent of S1(Magalhaes-Castro and Saraiva, 1971; Beck et al., 1996), while inthe short-tailed opossum (Monodelphis domestica), movementscould only be elicited when stimulating the face representation ofS1 (Frost et al., 2000). This partial motor map (face but not bodyrepresentation) of the short-tailed opossum is in direct contrastto the results observed in the big-eared opossum and the Virginiaopossum. While thalamic connectivity of S1 has been investi-gated previously in Virginia opossums, adding further supportfor the existence of an entirely overlapping sensorimotor amal-gam (Killackey and Ebner, 1973; Donoghue and Ebner, 1981a,b;Foster et al., 1981); to date there has been no study of the thalamicprojections to S1 and the surrounding areas in the short-tailedopossum. Thus, the primary aim of the current investigation isto characterize the thalamic projections to numerous somatosen-sory and surrounding cortical areas of the short-tailed opossumneocortex.

In previous studies, marsupials (including the short-tailedopossum) have been shown to have at least two complete soma-totopic representations within the neocortex: S1 and the secondsomatosensory area, S2 (Beck et al., 1996; Huffman et al., 1999b;Catania et al., 2000; Frost et al., 2000; for review see Karlen andKrubitzer, 2007). Further analysis of electrophysiological record-ings, architecture, and connectional patterns by our own andother laboratories has supported the presence of additional fieldsassociated with somatosensory processing, including a rostralfield (SR) as well as a caudal field (SC; Beck et al., 1996; Elstonand Manger, 1999; Huffman et al., 1999b; Wong and Kaas, 2009;Anomal et al., 2011; Dooley et al., 2013). A third complete soma-totopic representation (the parietal ventral area, PV) has beenidentified in numerous marsupials investigated (Beck et al., 1996;Elston and Manger, 1999; Huffman et al., 1999b), however despitecareful exploration, a separate, third somatotopic representationhas not been identified in the short-tailed opossum (Catania et al.,2000; Frost et al., 2000). However, the region which has beenidentified shows characteristics of both S2 and PV, thus we haveelected to refer to this area as S2/PV throughout the text. Further,a previous study conducted by our laboratory in the short-tailedopossum identified a multimodal region (MM), caudal to SC androstral to V2, which had connections with both somatosensoryand visual areas of the neocortex (Dooley et al., 2013).

In the present investigation we examined and quantified thethalamic projections to three somatosensory fields in the short-tailed opossum (SR, S1 and SC), as well as the multimodal region(MM). Further, in order to investigate whether different body partrepresentations within S1 were associated with the motor system,thalamic projections restricted to the body or the face representa-tion of S1 were investigated separately and in some cases directlycompared. To determine patterns of thalamocortical connectivity,several retrograde anatomical tracers were injected into architec-tonically and/or electrophysiologically defined locations withinthese fields and were related to the patterns of projections fromarchitectonically defined thalamic nuclei.

METHODSNeuroanatomical tracer injections were combined with architec-tonic analysis in eight adult short-tailed opossums (4 males, 4females, 76–136 grams) to determine the thalamocortical con-nections of parietal cortical areas, including both the lateral andmedial portions of primary somatosensory cortex (S1, for abbre-viations see Table 1), the rostral and caudal somatosensory fields(SR and SC respectively), and the cortical region just caudal to SC(multimodal cortex, or MM). In several cases, electrophysiologi-cal recordings were performed to identify the receptive fields forneurons at or surrounding the injection site (for table of injectionsites and cases see Table 2). All animals were housed in standardlaboratory conditions, with food and water available ad libitum.Animals were maintained on either a 12-h light/dark cycle ora 14/10 h light/dark cycle. All protocols were approved by theInstitutional Animal Care and Use Committee of the University ofCalifornia, Davis, and all experiments were performed under theNational Institutes of Health’s guidelines for the care of animalsin research.

NEUROANATOMICAL TRACER INJECTIONSAnimals were placed in an induction chamber and anesthetizedwith isoflurane (1–5% per liter O2). Following induction, the ani-mal’s head was shaved and a specially fitted mask was placed overthe animal’s nose. Throughout the procedure, the surgical planeof anesthesia was maintained with 1–3% isoflurane. A 2% lido-caine solution was injected subcutaneously at the midline of thescalp and around the ears, and animals were paced in a stereotaxicapparatus. Animals were given dexamethasone (0.4–2.0 mg/kg,IM) and atropine (0.04 mg/kg, IM) at the start of the surgery.Respiration and body temperature were monitored throughoutthe procedure.

Under standard sterile conditions, an incision was made atthe midline of the scalp, the temporal muscle was unilaterallyretracted, and a small craniotomy was performed over parietalcortex. In some cases, a photograph was taken over the exposedcortex to record the position of the tracer injections relativeto blood vessels. In several cases, electrophysiological record-ings were performed to confirm the placement of the injections(Table 2). A small hole was made in the dura over the area ofthe injection site, and either a custom-beveled Hamilton syringe(Hamilton Co., Reno, NV) or a Hamilton syringe fitted with aglass pipette beveled to a fine tip was lowered ∼300–400 µminto the cortex. Between 0.15 and 0.3 µL of 10% Fluoro-emerald






Table 1 | List of abbreviations used throughout the text.

CORTICAL FIELDS

3a Somatosensory area (deep)

A1 Primary auditory cortex

EC Entorhinal cortex

FM Frontal myelinated area

M1 Primary motor cortex

MM Multimodal cortex

PP/PPC Posterior parietal cortex

S1 Primary somatosensory area

S1 face Primary somatosensory area, face division

S1 body Primary somatosensory area, body division

S2 Second somatosensory area

SC Somatosensory caudal area

SR Somatosensory rostral area

V1 Primary visual area

V2 Second visual area

SUBCORTICAL STRUCTURES

APT Anterior pretectum

CeM Central medial nucleus

CL Central lateral nucleus

CP Cerebral peduncle

eml External medullary lamina

Hb Habenula

iml Internal medullary lamina

LGNd Lateral geniculate nucleus, dorsal division

LGNv Lateral geniculate nucleus, ventral division

MD Medial dorsal nucleus

MGNd Medial geniculate nucleus, dorsal division

MGNm Medial geniculate nucleus, magnocellular division

MGNv Medial geniculate nucleus, ventral division

mt Mammillothalamic tract

MV Medioventral nucleus

ot Optic Tract

PAG Periaqueductal gray

pc Posterior commissure

Pol Posterior nucleus (lateral)

Pom Posterior nucleus (medial)

Pul Pulvinar

PV Paraventricular nucleus

Rt Reticular nucleus

ST Subthalamic nucleus

VA Ventral anterior nucleus

VB Ventral basal nucleusVL Ventrolateral complexVM Ventromedial nucleusVMb Ventral medial basal nucleusVP Ventral posterior nucleusVPl Ventral posterolateral nucleusVPm Ventral posteromedial nucleusOTHER ABBREVIATIONS

CO Cytochrome oxidaseCTB Cholera toxin subunit-BFE Fluoro-emeraldfl forelimbFR Fluoro-rubyvGluT2 Vesicular glutamate transporter 2

(FE, Invitrogen, Carlsbad, CA), Fluoro-ruby (FR, Invitrogen,Carlsbad, CA), or cholera toxin subunit-B (CTB; Sigma-Aldrich,St. Louis, MO) was pressure-injected into the cortex. A total of12 tracer injections were made in 8 animals with several animalsreceiving multiple injections (Table 2). Following the injection,the surface of the brain was flushed with sterile saline to removeany remaining tracer on the cortical surface, the craniotomy wascovered with either bone wax or an acrylic skull cap (dependingon the size of the opening), and the temporal muscle and skinwere sutured. Antibiotics (Baytril, 5 mg/kg, IM) and analgesics(buprenorphine, 0.03 mg/kg, IM) were given. Animals recov-ered for 5–7 days to allow for the transport of the tracer, afterwhich they were euthanized with an overdose of sodium pen-tobarbital (Beuthanasia; 250 mg/kg, IP), transcardially perfusedwith 0.9% saline, followed by 2–4% paraformaldehyde in phos-phate buffer (pH 7.4), and finally 2–4% paraformaldehyde in10% phosphate-buffered sucrose. These procedures have beendescribed previously (Dooley et al., 2013).

ELECTROPHYSIOLOGICAL RECORDINGSIn three cases (08-80, 09-32, and 12-18) electrophysiologicalrecording experiments were performed following 5–7 days ofrecovery. When possible, these experiments helped confirm theS1/S2 boundary, the boundary of the face/body representa-tions within S1, as well as the rostral and caudal extent ofS1 (Figures 1B,C). For terminal electrophysiological recordingexperiments, two animals were anesthetized with 1–3% isofluraneand one was anesthetized using 30% urethane in propylene gly-col (1.25 g/kg, IP). No differences were noted between maps usingdifferent anesthesia methods. Surgical procedures were the sameas those described previously, except a larger craniotomy was per-formed over the entire neocortex and the dura was retracted.Digital images were taken so that electrophysiological recordingsites could be related to injection sites and cortical vasculature.

Multiunit electrophysiological recordings of parietal cortexwere performed using tungsten microelectrodes (0.010 inches,0.5–5 M�; A-M Systems, Sequim, WA). The electrode was low-ered ∼400 µm below the pial surface, in layer IV of cortex.Multiunit activity was amplified and filtered (100–5,000 Hz; A-MSystems Model 1800 Microelectrode AC Amplifier; A-M Systems,Carlsborg, WA), monitored through a loudspeaker, and viewedon a computer monitor. At each recording site neural responsesto somatosensory stimulation (consisting of light taps, displace-ment of vibrissae, brushing of skin, hard taps and manipulation ofmuscles and joints) were recorded and receptive fields were bothdrawn on illustrations of the body and documented in surgicalnotes. These methods have been previously described (Dooleyet al., 2013). Following electrophysiological recording, animalswere euthanized and perfused as described above.

HISTOLOGYOnce perfused, the brain was extracted, weighed, and pho-tographed and the cortex was separated from the subcorticalstructures. In some cases, the hippocampus and basal gangliawere dissected from the cortical hemispheres. All dissected cor-tices were then manually flattened between glass slides, brieflypost-fixed in 4% paraformaldehyde in 10% phosphate-buffered






Table 2 | Asterisks next to case numbers indicate cases for which corticocortical connections were reported previously (Dooley et al., 2013).

Case Area injected + halo Hemisphere injected Tracer Volume injected (µl) Injection area (mm2) Injection RF Figure number

13-114 S1 body Left FR 0.2 0.07 6

12-13* S1 body Right FR 0.3 0.11 7

13-126 S1 face Left FR 0.2 0.07 Face/Vib 2, 8

09-18* S1 face Left FR 0.3 0.07

12-18* S1 Right FR 0.3 0.06 Vib/Forepaw 9

13-73 S1 Right FE 0.3 0.07

08-29* SC Left FE 0.3 0.08 10

09-18* SC Left FE 0.3 0.14

13-73 SC Right FR 0.2 0.12 11

08-80* MM Left FE 0.3 0.07 Unresponsive 12

08-80* SR Left FR 0.3 0.07 Lips 13

13-126 S1 body, SR Left CTB 0.15 1.03 Forepaw 2

Receptive field of injection site is shown, when available.

sucrose, then allowed to soak for 12–36 h in 30% phosphate-buffered sucrose. The flattened cortex was then sectioned at30 µm using a freezing microtome. Alternating cortical sectionswere stained for myelin (Gallyas, 1979) or mounted immediatelyfor fluorescent microscopy. In cases in which CTB was injected,cortical sections were divided into three series, one of which wasreacted for CTB.

Subcortical structures were post-fixed as described above andthen allowed to soak in 30% phosphate-buffered sucrose untilthey sunk in solution (24–48 h). Following this, they were sec-tioned coronally at 30–40 µm. Tissue was divided into 3 or 4series. One series in all cases was stained for cytochrome oxidase(CO; Wong-Riley, 1979) while a second was mounted imme-diately for fluorescent microscopy. When applicable, immuno-histochemistry was performed for CTB. Additionally, all caseswere stained for either Nissl or processed for vGluT2 expression(mouse monoclonal anti-vGluT2 from Millipore, Billerica, MA;1:5000).

DATA ANALYSISInjections sites and cortical field boundaries were reconstructedas described previously (Campi et al., 2010; Dooley et al., 2013).Briefly, reconstructions of anatomical boundaries from pho-tographs of individual myelin sections were made (Figure 1A),and the boundaries from the entire series were examined andcombined into a single reconstruction (Figure 2A; for details,see Seelke et al., 2012). Each reconstructed section containedan outline of the section, blood vessels, tissue artifacts, probes,and architectonic borders of cortical fields. These landmarkswere then directly related to sections mounted for fluores-cence or processed for CTB, and a comprehensive reconstructionof the neocortex was generated that contained the injectionsite, injection halo, labeled cells and architectonic boundaries.Corticocortical connections for these cases have been describedpreviously (Dooley et al., 2013). Several injections were foundto have spread into surrounding cortical areas, and thus thesecases were not included in quantitative analysis and Table 2;however the data were qualitatively described and illustrated asfigures (e.g., Figure 2). In all cases included in the study, cortical

injections spanned all cortical layers and did not extend into theunderlying white matter (Figures 1D–H).

For thalamic sections, labeled cells were plotted using anX/Y stage encoding system (MD Plot, Minnesota Datametrics,St. Paul, MN) mounted to a fluorescent microscope and con-nected to a computer. Additionally, the tissue outline, bloodvessels, and artifacts were plotted, which allowed these sectionsto be aligned to the anatomical boundaries determined from theNissl, CO or vGluT2 stained tissue (Figures 2C,D). The loca-tion of labeled cells in the plotted sections was combined withadjacent sections to form a single comprehensive reconstruc-tion (Figure 2B). When necessary, brightness and contrast wereadjusted for the digital images using Photoshop CS5 (Adobe,San Jose, CA). Additionally, in several instances, multiple pho-tographs of a single cortical or thalamic section were turned into asingle composite image using Microsoft Image Composite Editor(Microsoft, Redmond, WA).

Thalamocortical connections were quantified by summing thetotal number of retrogradely labeled cells in the thalamus andcalculating the percentage of labeled cells in a given thalamicnucleus. This allowed us to normalize the data for injections ofdifferent sizes. This quantification allowed us to determine con-nection strength for each thalamic nucleus as follows: Strong: >

10%; Moderate: 9% to 3%; Weak: 3% to 1%; Intermittent: < 3%and not present in all cases.

RESULTSThe goal of these studies was to determine the thalamocorticalconnections of parietal somatosensory areas as well as the mul-timodal region in the short-tailed opossum; with the ultimategoal of comparing these connections with other marsupials aswell as with eutherian mammals. In the following results we firstdescribe how cortical field boundaries were determined and theextent to which our injection sites were restricted to the differ-ent areas of interest. Next, we define the different nuclei in thethalamus by their appearance in histologically processed tissue.Finally, we describe the patterns of thalamocortical connectionsof S1, SC, SR, and MM and quantify the density of projectionsfrom different thalamic nuclei.






FIGURE 1 | Cortical organization in Monodelphis domestica. (A) Atangential section of cortex that has been stained for myelin. The medial wall,caudal wall and piriform cortex have been removed. The arrow points to aninjection located in SC. (B) An illustration of the body of the short-tailedopossum colored according to the head representation (dark green) and thebody representation (light green). (C) Schematic of cortical fields and bodyrepresentations in a brain oriented as in A. Different cortical fields are depictedin different colors; these colors are used in later figures showing theanatomical connections (labeled cell bodies in the thalamus) to indicate thecortical field location of the injection site. Thick lines delineate bordersbetween different cortical areas, while thin lines separate different body part

representations within S1 and S2/PV determined in previouselectrophysiological recording studies. Boundaries of cortical areas are basedon architectural, connectional, and electrophysiological data, whiletopographic boundaries within S1 are based on electrophysiologicalexperiments. The functional organization of S1 and S2/PV is adapted fromCatania et al. (2000). (D–H) Superficial to deep sections of the corticalinjection in A. Other than being slightly larger in the most superficial section(D), the injection halo remains similar in size through intermediate sections(E–G), before it finally begins to shrink in the final section (H). As with all othercases included in this study, the injection halo included all cortical layers butdid not extend into the underlying white matter. See Table 1 for abbreviations.

DETERMINATION OF CORTICAL FIELD BOUNDARIESA subset of the cases used in this study were also used in ourprevious study of corticocortical connections of parietal cortexin Monodelphis domestica (Table 2; Dooley et al., 2013). In addi-tion to our previous publication, more extensive descriptions ofthe organization of different cortical fields in this species canbe found in other studies by our own and other laboratories(Huffman et al., 1999b; Catania et al., 2000; Frost et al., 2000;Kahn et al., 2000a; Karlen et al., 2006; Karlen and Krubitzer,2009; Wong and Kaas, 2009). In many of these studies, myelinstains were directly compared to electrophysiologically identifiedboundaries (Catania et al., 2000). Additionally, the neocortex ofshort-tailed opossums has been assessed in sagittal and coronalsections, demonstrating the laminar distribution of myelin and

CO (Wong and Kaas, 2009). As in the present study, the primarysensory areas of the short-tailed opossum, including S1, V1, andA1, were densely myelinated and their borders could be readilydetermined (Figure 1A, see Table 1 for abbreviations). SR andSC were lightly myelinated strips of cortex approximately 0.5 mmwide and were located directly rostral and caudal to S1 respec-tively (Figure 1). V2 was identified as a lightly myelinated stripapproximately 0.5 mm at the rostral boundary of V1. Multimodalcortex (MM) was a very lightly myelinated region caudal to SCand rostral to V2. S1 contains a representation of the entirecontralateral body surface, with the hindpaw represented mostmedially and the rhinarium and oral structures represented mostlaterally (Figures 1B,C; Catania et al., 2000). Thus, S1 can be fur-ther divided functionally into body (S1 body) and face (S1 face)






FIGURE 2 | VPm and VPl project to different body representations within

S1. (A) Reconstruction of the flattened cortical hemisphere from case 13-126in which one injection of CTB (light green circle) was placed in the S1 bodyrepresentation and one injection of FR was placed in the S1 facerepresentation (dark green circle). (B) Reconstructed thalamic section showingthe boundaries of different subcortical nuclei drawn from histologicallyprocessed tissue. Dorsal is up, lateral is to the left. Colored dots and squarescorrespond to labeled neuronal nuclei resulting from each injection, with lightgreen circles projecting to the light green S1 body injection and the dark green

squares projecting to the S1 face injection in (A). Dashed lines indicateborders that were more difficult to determine from the sections available. Thegray box corresponds to the region of the light field and fluorescent images in(C–F). Light field image of tissue stained for (C) vGluT2, (D) CO, (E) CTB, and(F) unstained tissue visualized under fluorescence microscopy. The arrows ineach box indicate the presence of blood vessels use to align tissue. The scalebar for (C–F) are all 500 µm. The boxes in (E) and (F) are magnified in (G) and(H), respectively, showing (G) neurons labeled with CTB and (H) FR. The scalebar for (G,H) is 100 µm. See Table 1 for abbreviations.

divisions, generally separated by a small band of lightly myeli-nated cortex. These techniques were utilized in S1 injections in thepresent study in order to determine the location of our injectionsites relative to the body or face representations.

ARCHITECTONIC SUBDIVISIONS OF THALAMUSSubdivisions of the thalamus were delineated using sectionsstained for CO and either Nissl substance or vGluT2, and manyof the nuclei described here have been previously distinguishedby our own and other laboratories (see Turlejski et al., 1994;Huffman et al., 1999b; Karlen et al., 2006; Olkowicz et al., 2008).The boundaries of thalamic nuclei in short-tailed opossumswere also compared to previously published thalamic boundariesreported in the closely related Virginia opossum (Oswaldo-Cruzand Rocha-Miranda, 1968; Jones, 2007).

In the rostral-most sections where label was found, numerousmidline nuclei were identified. CeM was located at the midline,was darkly stained, and had densely packed neurons. It was mostapparent in Nissl-stained tissue, but was also visible in CO andvGluT2 preparations (Figures 3A–D). Dorsal and lateral to CeM

lies another darkly stained nucleus termed CL (which is partof the intralaminar group; Figures 3A–D). As with CeM, CL ismost apparent in Nissl-stained tissue but can also be seen intissue stained for CO and vGluT2. Medial to CL and dorsal toCeM is a large oval nucleus, MD, that has moderate neuronalpacking density in Nissl-stained sections (Figure 3B), and stainslightly for both CO and vGluT2 (Figures 3A,C). Boundaries sep-arating these nuclei can be readily determined by the sharpcontrast between the darkly stained CeM and CL and the lightlystained MD.

Lateral to these midline nuclei in more rostral portions ofthe thalamus we observed labeled neurons in the ventral ante-rior and the ventrolateral nuclei (VA and VL, respectively).The boundaries between these two nuclei were not always dis-tinct in CO and Nissl-stained tissue, and so throughout thequantitative analysis performed in these studies they were com-bined into a single category (“VL/VA”). Both of these nucleistained moderately for CO and showed a medium neuronalpacking density in Nissl stains (Figures 3A,B). When tissuewas stained for vGluT2, VA stained more darkly than VL and






FIGURE 3 | Boundaries of nuclei in rostral sections of the dorsal

thalamus in the short-tailed opossum. Three different stains were usedto delineate the boundaries of thalamic nuclei including CO (A) Nissl(B) and vGluT2 (C). Reconstructions of thalamic nuclei derived from thesestains are drawn in (D). Midline nuclei, including CeM, CL and MD, can beobserved using each stain, but are most obvious in Nissl and vGluT2.Ventral and lateral to midline nuclei, VL, VA, and VM can also be seen. VAand VM boundaries are most apparent in vGluT2 (C). Lateral to thesestructures, LGNd and LGNv can be identified. The boundaries of thesestructures can be readily identified in CO (A) and vGluT2 (C) stained tissue.In all images, dorsal is up and lateral is to the left. Scale bar in (A) is500 µm, and is the same for all images.

was located medial and ventral to VL (Figure 3C, boundarydenoted by the dotted line in Figure 3D). Just medial to VAwas VM, a densely packed nucleus that stained darker thanthe surrounding nuclei in Nissl-stained sections (Figure 3B).However, VM stained moderately for CO and lightly for vGluT2(Figures 3A,C). VM extended further caudal than VL and VA(Figures 4A–D).

The reticular nucleus, Rt, could be identified throughout mostof the rostrocaudal extent of the thalamus (e.g., Figures 3, 4).Rt was heterogeneous in appearance due to the number of fiberbundles passing throughout this nucleus. Despite its heteroge-neous appearance, Rt stained darker then the surrounding tissuein all stains, and appeared particularly dark in tissue stained forvGluT2 (Figures 3C, 4C). Throughout most of its rostral-caudalextent, Rt was bounded dorsomedially by the external medullarylamina (eml), which was clearly visible in both CO and vGluT2stained tissue as a very lightly stained structure (see Figures 3A,C,4A,C).

The ventral posterior nucleus (VP) was located caudal to VLand VA. It is bordered ventrally by iml and Rt and laterally byeml. VP stained darkly for CO, Nissl and vGluT2 (Figures 4A–C).VP could be further subdivided into lateral (VPl) and medial(VPm) divisions, with VPl corresponding to the representationof the body and VPm corresponding to the representation ofthe face. Further, VPm has been described as having a higher

FIGURE 4 | Boundaries of nuclei in the dorsal thalamus at the level of

the ventral posterior nucleus in the short-tailed opossum. As in theprevious figure, three different stains were used to delineate boundaries;CO (A), Nissl (B), and vGluT2 (C), with reconstructions derived from thesestains illustrated in (D). The ventral posterior complex, including VPm andVPl can be seen as a darkly staining region in CO (A) and vGluT2 (C) stainedtissue, with VPm staining more darkly. VMb can be seen medial to VP as alightly stained line of tissue between two darkly stained sections. Lateral toVP, eml stains lightly in all three series. At this level of the thalamus, Pol isintermingled with eml. As in Figure 3, LGNd and LGNv are lateral to theventral posterior complex, and are most apparent in CO (A) and vGluT2(C) stained tissue. Near the midline, PF can be seen in CO (A) and vGluT2(C) stained tissue, being slightly darker than the surrounding nuclei. Finally,dorsal to VMb and medial to VP, the caudal extent of VM could be identifiedas a lightly stained nuclei in all three series. Conventions as in Figure 3.

density of cells compared to VPl, and thus appeared darkerthan surrounding tissue in a Nissl stain, and stained darker forCO. The boundary between VPl and VPm was most appar-ent in tissue stained for CO (see Figure 4A). Dorsal, caudal,and in some sections lateral to VP was the posterior nucleuscomposed of both a medial (Pom) and lateral (Pol) division.Pom extended rostrally and is located just medial to VP, stain-ing lightly for both CO and Nissl (Figures 4A,B). Medial toVP we identified the ventral medial basal nucleus (VMb). Thisnucleus stained moderately for CO and Nissl, and often appearedlighter than regions immediately dorsal, ventral and medial to it(Figures 4A,B). In tissue stained for vGluT2, VMb stained darkly(Figure 3C).

As has been described previously (Huffman et al., 1999a;Karlen et al., 2006), in Monodelphis both LGNv and LGNd staineddarkly for CO, and are separated from each other by a thin lightlystained region (see Figures 3A, 4A). LGNv contained a lateralportion that stained very darkly for both CO and in Nissl. LGNv






extended medially to the lateral border of Rt (Figures 3A–D,4A–D). In tissue stained for vGluT2, LGNd stained much moredarkly than the surrounding tissue, and individual laminae couldbe observed (Figures 3C, 4C). Dorsomedial to the LGNd is thepulvinar (Pul, previously termed the lateral posterior thalamicnuclei; Kahn et al., 2000b). Neurons in Pul showed a moderatepacking density in tissue stained for Nissl and CO (Figures 4A,B).Pul was moderately stained by vGluT2, and the caudal sub-division stained more intensely near the dorsal-most extent(Figure 4C; Baldwin et al., 2013). This subdivision is indicatedwith a dotted line in Figure 4D.

In the caudal most portions of the thalamus, Pol could beidentified. In the rostral sections where it was identified, Pol wasmostly intermingled with eml (see Figures 4A–C, 5A–C, 6D–G).While the boundaries of Pol were at times difficult to identifywith the stains used in the present studies, in favorable sectionsit could be identified as a lightly staining nucleus lateral to VP butmedial to LGN. In the more rostral sections Pol is bordered ven-trally by Rt. Finally, the MGN began to emerge just caudal andlateral to VP. In CO and vGluT2 stained tissue, fiber bundles run-ning perpendicular to the tissue could be seen separating MGNfrom VP (Figures 5A–C, 6H). As the MGN begins to emerge,the region surrounding and between these nuclei was identifiedas the caudal extension of Pol described above (Figures 5A–C,6H). In more caudal sections of the thalamus MGN becamelarger and was located further laterally, and could be furthersubdivided into different divisions, including the darkly stainedMGNv as well as the more lightly staining MGNd and MGNm(Figure 6I).

FIGURE 5 | Boundaries of nuclei in the dorsal thalamus at the

caudal-most extent of the ventral posterior nucleus in the short-tailed

opossum. All three stains used are the same in Figures 3, 4, along with anillustration of nuclear boundaries. In these sections VP, Pol and MG can beidentified. Pol is lateral to VP and extends outward, surrounding theemerging MGN. Conventions as in previous two figures.

THALAMIC CONNECTIONS OF THE PRIMARY SOMATOSENSORY AREA(S1)Of the six cases in which injections were made in S1, two ofthe injections were restricted to the body representation of S1(Figures 6, 7), two were restricted to the face representation of S1(Figure 8; case 09-18, not shown), and two were placed across theS1face/S1body representations (Figure 9; case 13-73, not shown).In all 6 cases, the majority of projections originated from VP(mean = 57.5%, see Table 3 for the percentages from individ-ual cases), with the percentage of labeled cells that originatedfrom VPm and VPl varying dramatically depending on the loca-tion of the cortical injection within S1. In one case, the injectionwas placed in the medial portion of S1 (in the expected repre-sentation of the forepaw, case 13-114, Figure 6A), resulting inthe vast majority of labeled cells residing in VPl (Figures 6D–I).Conversely, a cortical injection placed in the lateral portion of S1(Figure 8A, confirmed with electrophysiological recording to berestricted to the vibrissae representation of the face) resulted inthe vast majority of labeled cells located in VPm (Figures 8D–H).In one case, two injections were placed such that one wasrestricted to S1 body representation and another was restrictedto S1 face representation (extending rostrally into SR, Figure 2A).In this case, VP neurons projecting to S1 face representation (darkgreen squares) are restricted to VPm while neurons projecting toS1 body representation (light green circles) are almost entirelyrestricted to VPl (Figure 2B). In nearly all cases in which S1 wasinjected, labeled cells were not spatially restricted to a particularportion of VPl or VPm, but were found throughout each of thesesubdivisions suggesting a rather loose topographic organizationof these nuclear subdivisions (e.g., Figures 2B, 7G).

In addition to projections from VP, all injections in S1 resultedin strong projections from Pom (mean = 18.6%) and moderateprojections from VM and VMb (mean = 4.9 and 6.0% respec-tively). For both of these nuclei we observed a difference in thepercentage of labeled neurons depending on the placement of theinjection within S1. Injections restricted to the body representa-tion in S1 did not have projections from VM, while all injectionsinto the face representation of S1 had moderate to strong projec-tions from VM (range = 2.8–12.2%). Projections from VMb to S1were not present in every case, but did not appear to differ system-atically for body versus face representations in S1 (see Table 3).However, the two injections with strong projections from VMbare located rostrally within S1, closer to the expected location ofthe representation of oral structures (Figure 6A; see Figure 1 forlocation of oral representation). Moderate to weak projectionswere also observed from PF and VA/VL, while all other nucleiin which labeled cells were found had weak and inconsistentprojections to S1 (e.g., CL, CeM, see Table 3).

THALAMIC CONNECTIONS OF THE CAUDAL SOMATOSENSORY AREA(SC)The three cases in which injections were restricted to SC hadvery consistent patterns of thalamic connectivity (Figures 10, 11,case 08-29, not shown). As with S1, the strongest projectionsoriginated from VP (mean = 39.3%), however SC also receivedstrong projections from Pol (M = 19.4%), VA/VL (mean =11.6%), and Pom (mean = 9.7%). These strong projections from






FIGURE 6 | Projections to the S1 body representation from in case

13-114. (A) Reconstruction of the flattened cortical hemisphere, showingthe core of the injection site of FR (black dot) surrounded by the halo(green circle) in the body representation of S1. (B) Fluorescent image ofthe FR injection site and surrounding halo. (C) Percent of labeled

neurons originating from various thalamic nuclei (D–I) Rostral to caudalprogression of CO stained tissue; corresponding thalamic borders andlabeled neurons are shown below (D′–I′). In this case, the majority oflabel was found within VPl, VMb, and Pom. All conventions as inprevious figures.

VL/VA were not spatially restricted, and were located through-out these nuclei (Figures 10C,D). Additionally, labeled neuronsin Pol were observed throughout the nucleus (Figures 10E–G,11H–L). Moderate projections to SC also originated from VM(mean = 6.0%). Notably, weak projections from LGNd and Pul(two visual nuclei) were also present in all three cases (mean =1.4 and 2.8% respectively). When comparing the thalamocorti-cal projection profiles of S1 and SC, the most notable differencesare the density of projections of VP, Pol and VA/VL (Table 3). S1had more dense projections from VP while SC had more denseprojections from Pol and VA/VL. Further, SC had weak but con-sistent projections from visual nuclei of the thalamus which werenot observed for S1 projections.

THALAMIC CONNECTIONS OF THE MULTIMODAL REGION (MM) ANDTHE ROSTRAL SOMATOSENSORY AREAS (SR)One injection was entirely restricted to MM (Figure 12A). In thiscase, MM received strong projections from VP and Pol (26.8 and27.7% respectively, see Figures 10F,G). Additionally, MM also

received significant projections from VL/VA (12.7%) and mod-erate projections from both LGNd and the pulvinar (6.6% forboth nuclei). Thus, MM is distinguished from SC by its rel-atively dense projections from LGNd and the pulvinar, givingMM a more multimodal connectional profile compared to theprimarily somatosensory SC, Additionally, we had one injectionentirely restricted to SR (Figure 13). Unlike other cortical parietalareas described thus far, SR received substantially more projec-tions from both VM (20.6%) and Pom (20.1%) than from VP(10.3%, see Figures 13D–H). SR also received very dense pro-jections from several midline structures that do not consistentlyproject to S1, SC, or MM. For example, SR received strong pro-jections from MD (16.1%), while VA/VL (8.9%), VMb (8.4%),and PF (5.5%) all have moderate projections to SR. Weak pro-jections were also observed from CL and CeM (1.1 and 1.2%,respectively).

Taken together, these data demonstrate that S1, SC, and MMreceived the strongest projections from VP; however the strengthof those projections decreased from S1 to SC and MM (see






FIGURE 7 | Projections to the S1 body representation in case

12-13. (A) Reconstruction of the FR injection into the bodyrepresentation of S1. (B) Fluorescent image of the FR injection siteand surrounding halo. (C, D) Distribution of labeled neurons wereobserved in similar nuclei to those shown in Figure 4, but were

also located in more rostral portions of the thalamus in VL/VA. (E–H)Labeled neurons throughout VPl and Pom have a similar distributionthat was illustrated in the previous figure. (I) Percent of labeledneurons originating from various thalamic nuclei. All conventions as inprevious figures.

Figure 14, Table 3). Conversely, MM had stronger projectionsfrom visual structures (LGNd and Pul) while SC has only sparseconnections from these nuclei and S1 had no projections fromthese nuclei. Mimicking the pattern of VP connections, thestrength of projections from Pom was weaker as injections movedfarther caudal in cortex (e.g., from S1 to SC to MM). Strongprojections from Pol were only found in areas caudal to S1(Figure 14F). Located immediately rostral to S1, SR showed a pat-tern of projections from thalamic nuclei that was notably differentfrom the other three areas described. Strong projections to SRwere seen from midline structures including VM and MD, whichonly weakly or inconsistently projected to the other cortical areasexamined (Figures 14B,F).

DISCUSSIONThe present investigation is part of a broader objective of our lab-oratory to appreciate the cortical organization and connectivityof early mammals, and how this basic plan was modified in dif-ferent lineages to produce the remarkable variability in behavior

observed in extant species. Comparative studies in general allowus to infer aspects of organization of our early ancestors; how-ever, some species can provide a more accurate representationof early mammals based on their phylogenetic status, lifestyle,as well as brain and body morphology (Kemp, 2005; Kaas, 2011;Rowe et al., 2011). One such species is the marsupial Monodelphisdomestica, or the short-tailed opossum. Previously, we exam-ined the corticocortical connections of several somatosensoryareas in Monodelphis and found evidence for three somatosen-sory fields (SR, S1, and SC) as well as a multimodal region(MM; Dooley et al., 2013). In the present experiments we inves-tigated the thalamocortical projections of these cortical fieldsand demonstrate that each cortical field has a unique pattern ofthalamocortical connections, supporting our parcellation of cor-tex. Additionally, we demonstrate different thalamic projectionsto the S1 body representation and the S1 face representation,which we interpret in the context of sensorimotor integrationfor specializations of the face versus the body in Monodelphisdomestica. Finally, thalamocortical connections to all cortical






FIGURE 8 | Projections to the face representation in case 13-126.

(A) The location of an FR injection site in the lateral portion of S1 inthe representation of the face. (B) Fluorescent image of the FRinjection site and surrounding halo. (C–H) Progression of the thalamic

sections with labeled neurons, showing labeled neurons largelyrestricted to VPm, Pom and VM. (H) Percent of labeled thalamicneurons in this experiment. (I) Percent of labeled neurons originatingfrom various thalamic nuclei. Conventions as in previous figures.

areas examined in the present study are compared to projectionsin other marsupials and in other mammals to better appreci-ate common patterns of this thalamocortical network as well asderivations.

THALAMIC PROJECTIONS TO S1 IN MARSUPIALSEarly studies in marsupials such as the Virginia opossum demon-strated that unlike placental mammals, marsupials do not appearto have a separate cortical motor area. Rather, they possess whathas been called a “somatosensory-motor amalgam” (Lende, 1963;Killackey and Ebner, 1973). The somatosensory-motor amalgamrefers to the complete overlap of the Virginia opossum’s topo-graphic representation of cutaneous receptors of the body andface in the primary somatosensory area (S1) with motor mapsof the body in the primary motor area (M1). Given that marsu-pials represent a very early mammalian radiation whose ancestorsdiverged over 150 million years ago (Nilsson et al., 2010), one cur-rent hypothesis is that their sensory motor amalgam represents

a primitive form of mammalian neocortex, which ultimatelysegregated into separate sensory and motor areas in placentalmammals (Kaas, 2004). While our understanding of the relation-ship between sensory and motor cortex has undergone enormoustransformations in the last decade (Hatsopoulos and Suminski,2011; Kaas et al., 2012), the initial observation of a complete lackof a separate motor cortex in marsupials has been validated inother marsupials including short-tailed opossums and the big-eared opossums (Magalhaes-Castro and Saraiva, 1971; Frost et al.,2000). Because it is likely that this type of organization doesindeed represent a more primitive form of cortical organization,determining the connectivity, particularly with somatosensoryand motor subcortical structures, is a critical step in understand-ing how the cortical motor system in placental mammals ulti-mately came to generate sophisticated motor control necessaryfor complex, goal-directed movements.

There are only a few studies of thalamocortical connectionsin marsupials. The thalamic projections to S1 in the Virginia






FIGURE 9 | Thalamic projections to the border of the face-body

representation in S1 in case 12-18. (A) Location of the FR injection site.(B) Fluorescent image of the FR injection site and surrounding halo. (C–F)

Labeled neurons can be seen throughout the medial and lateral divisions ofVP, along with labeled cells in Pom and VMb. (G) Percent of labeled thalamicneurons projecting to S1. Conventions as in previous figures.

Table 3 | Percentage of labeled neurons observed in different thalamic nuclei out of total labeled neurons in the thalamus.

Case Area injected VA/VL VM CL CeM MD VPm VPl VP VMb Pom Pol PF LGNd Pul Other

12-13 S1 body 6.0 0.0 0.0 0.0 0.0 0.0 72.0 72.0 1.0 18.0 0.0 0.0 0.0 0.0 3.0

13-114 S1 body 0.0 0.0 0.0 0.0 0.0 2.8 38.9 41.7 10.2 30.6 0.0 4.6 0.0 0.9 12.0

13-126 S1 face 2.3 12.2 3.8 0.8 3.8 52.7 3.1 55.7 0.8 12.2 0.0 5.3 0.0 0.0 3.1

09-18 S1 face 0.0 2.8 0.0 0.0 0.0 43.4 26.4 69.8 10.4 11.3 1.9 2.8 0.0 0.0 0.9

12-18 S1 0.0 3.0 0.0 0.0 0.0 13.4 36.6 50.0 9.7 22.4 1.5 7.5 0.0 0.0 6.0

13-73 S1 4.0 11.4 4.0 1.5 0.0 23.8 32.2 55.9 4.0 16.8 0.0 0.0 0.0 0.0 2.5

Average 2.0 4.9 1.3 0.4 0.6 22.7 34.9 57.5 6.0 18.6 0.6 3.4 0.0 0.2 4.6

SEM 1.0 2.2 0.8 0.3 0.6 8.8 9.1 4.7 1.9 2.9 0.4 1.2 0.0 0.2 1.6

13-73 SC 10.4 7.4 2.8 0.0 1.2 9.0 24.2 33.3 0.0 16.6 17.6 0.0 0.5 4.2 6.2

09-18 SC 3.5 1.2 0.0 0.0 0.0 11.0 44.1 55.1 1.2 2.8 26.4 0.0 2.4 2.4 5.1

08-29 SC 20.7 9.5 3.8 1.1 0.2 14.0 15.5 29.5 0.7 9.6 14.4 0.5 1.3 1.8 7.0

Average 11.6 6.0 2.2 0.4 0.4 11.4 27.9 39.3 0.6 9.7 19.4 0.2 1.4 2.8 6.1

SEM 5.0 2.5 1.1 0.4 0.4 1.5 8.5 8.0 0.3 4.0 3.6 0.2 0.5 0.7 0.6

08-80 MM 12.7 0.9 0.5 0.0 1.4 9.9 16.9 26.8 0.5 8.5 27.7 0.0 6.6 6.6 8.0

08-80 SR 8.9 20.6 1.1 1.2 16.1 7.4 2.9 10.3 8.4 20.1 0.0 5.5 0.0 0.1 7.7

opossum are from the VP as well as from VL/VA, nuclei associatedwith the motor system (Killackey and Ebner, 1973; Donoghue andEbner, 1981b; Beck et al., 1996). Projections from VPl and VPmproject to the body and face representations of S1, respectively,and a previous study in the big-eared opossum has describedeven finer somatotopy within VP (Sousa et al., 1971). Whilethe current investigation did not examine somatotopy at this

level of specificity, our results are consistent with this previousreport. Additionally, strong projections to S1 have also beenreported from the medial division of the posterior nucleus, Pom(Killackey and Ebner, 1973; Donoghue and Ebner, 1981b). Intwo Australian marsupials, brush-tailed possums and Northernquolls, cortical projections of VL and VP were overlapping, suchthat a notably large portion of parietal cortex (presumed to be






FIGURE 10 | Thalamic projections to area SC in case 08-29. (A) Aninjection of FE into SC which extends slightly into the caudal-most portion ofS1. (B–D) Densely packed labeled cells can be seen in VL/VA and VM, (D–G)

as well as throughout VPm and VPl. Large numbers of labeled cells can also

be seen throughout Pol, sometimes extending laterally into LGNd and Pul.(H) A small number of cells were seen projecting from the medial geniculatecomplex. (I) Percent of labeled thalamic neurons projecting to SC.Conventions as in previous figures.

S1) received projections from both nuclei (Haight and Neylon,1978a,b, 1981a,b). Notably, for projections from both VL and VP,thalamic projections corresponding to distal body parts (e.g., dig-its of the paws) projected to more rostral portions of cortex, andthalamic projections corresponding to proximate body parts (e.g.,the trunk) projected to more caudal portions of cortex. In con-trast, in placental mammals the topography of projections to cor-tex from VL are mirrored relative to those of VP, with distal bodyparts (e.g., digits of the paws) projecting to relatively caudal por-tions of cortex compared to proximate body parts. Unfortunately,neither electrophysiological nor histological techniques were usedto confirm the placement of injections into parietal cortex in theseAustralian marsupials, making thalamocortical projections fromVL and VP to S1 difficult to compare across species.

These results described in other marsupials are consistentwith those described in the present investigation in the short-tailed opossum with respect to VP and Pom. However, short-tailed opossums differ from other marsupials studied in thatthey do not have significant projections from VL/VA throughoutS1, with an average of only 2% of thalamic projections orig-inating from VL/VA, and half of S1 injections resulting in noprojections from this nucleus (Table 3). The lack/inconsistencyof projections from VL/VA to S1 does not appear to differbased on the somatotopic origin of the injection site, a findingthat is surprising considering previous results which demon-strate a difference in the motor movements elicited followingintracortical microstimulation (Frost et al., 2000; see below forfurther discussion).






FIGURE 11 | Thalamic projections to area SC in case 13-73.

(A) Location of cortical injection sight of FR into SC. (B–E) Labeled cellscan be seen throughout VL/VA and VM. (F–J) Additional numerouslabeled cells can be seen throughout VPl and VPm, as well as Pom.(K,L) As with Figure 8, labeled cells can be seen throughout Pol,

sometimes extending laterally into LGNd and Pul, along with labeled cellsat the caudal-most extent Pol as the Medial Geniculate complex beginsto emerge. (M) A single cell was observed in the medial geniculatecomplex. (N) Percent of labeled thalamic neurons projecting to SC.Conventions as in previous figures.

THALAMIC PROJECTIONS TO S1 IN OTHER MAMMALSStudies in monotremes, an order of mammals whose ancestorsradiated approximately 220 million years ago (Van Rheede et al.,2006), reveal the diversity of sensorimotor organization of earlymammals. There are only three extant species of monotremesand studies of cortical connections in these species are extremely

limited. Within the neocortex, the platypus and echidna havebeen found to have three somatosensory areas; S1, a rostral areatermed R, and a second somatosensory area, which may corre-spond to S2 or PV. Studies also suggest the existence of a motorarea rostral to R (Krubitzer et al., 1995). Thalamic projectionsto S1 have only been described in two studies in the echidna;






FIGURE 12 | Thalamic projections to multimodal region (MM) in case

08-80. (A) Injection site of FE in MM. (B) Fluorescent image of the FEinjection site and surrounding halo. (C–G) Labeled neurons can be seenprojecting to MM from large portions of VL/VA as well as from VPl and VPm.

Additionally, densely packed label neurons are observed in Pol; moderatenumbers of labeled neurons in LGNd and Pul. (H) Percent of labeled thalamicneurons projecting from various thalamic nuclei to MM. Conventions as inprevious figures.

one using degeneration methods (Welker and Lende, 1980) andone using retrograde tracers (Ulinski, 1984). Welker and Lende(1980) demonstrated that lesions to S1 resulted in degeneratedneurons throughout VP and in a nucleus dorsal and caudal toVP (possibly Pom). Ulinski (1984) described projections exclu-sively from VP to S1. Neither study describes projections fromVL to S1; however the delineation of thalamic nuclei in theechidna has undergone significant revisions since these studieswere performed (Mikula et al., 2008), and thus these early studies

may have failed to properly differentiate the VP/VL boundary.Regardless, these studies support the basic mammalian pattern ofprojections from VP to S1, and suggest projections from VP andVL do not overlap in cortex in the echidna.

There are numerous studies of thalamocortical projections toS1 in placental mammals, which report slight modifications onthe same basic plan of thalamocortical connectivity. Because acomplete review of this literature is beyond the scope of this dis-cussion, we will focus on the pattern of thalamic projections in






FIGURE 13 | Thalamic projections to SR in case 08-80.

(A) Reconstruction of the injection of FR in SR. (B) Fluorescent image ofthe FR injection site and surrounding halo. (C) Percent of labeled thalamic

neurons projecting to SR. (D–H) Unlike previous injections, the majority oflabel cells were in the midline nuclei, including PV, MD, and PF.Conventions as in previous figures.

small-brained placental mammals, such as rodents. Studies inrats, mice, squirrels, and naked-mole rats describe thalamic pro-jections to S1 from VP and Pom (Wise and Jones, 1977; Krubitzerand Kaas, 1987; Henry and Catania, 2006; Liao et al., 2010). Morerecently, it has been demonstrated in rats that projections to S1from the thalamus are not homogeneous; instead there are paral-lel pathways to different cortical layers as well as to the granularand dysgranular zones within S1. VP projects to the granularzones and Pom projects to the dysgranular zones (Liao et al., 2010;Viaene et al., 2011; Ohno et al., 2012). It has been suggested thatthese two thalamic pathways to cortex are functionally distinct,with VP driving activity in S1 and Pom having more of a modu-latory role (Hoogland et al., 1987; Liao et al., 2010; Viaene et al.,2011; Ohno et al., 2012). The present investigation confirms thatas in rodents, VP and Pom project to S1, but injections in thepresent study were not restricted to a particular layer of cortex andthere are no architectonically distinct granular and dysgranularzones in Monodelphis.

Weak and/or topographically restricted projections from VLand VM to S1 have also been described in some rodent species.VL projections to the forelimb and hindlimb representations inS1 have been demonstrated in rats; these representations dis-play properties of both S1 and M1 in this species (Donoghueet al., 1979; Cicirata et al., 1986; Aldes, 1988) and other rodents(Henry and Catania, 2006; Liao et al., 2010). Sparse projectionsfrom VM to S1 are reported in diverse rodent species rangingfrom rats to squirrels to naked mole rats (e.g., Donoghue et al.,

1979; Krubitzer and Kaas, 1987; Giannetti and Molinari, 2002;Henry and Catania, 2006), although these projections are oftenweak and not found in every case. In rats, VM has been shownto project primarily to rostral cortical areas, and more weakly toparietal cortex (Herkenham, 1979). Interestingly, in naked molerats, Henry and Catania (2006) describe weak projections fromVM to the S1 forepaw representation in every case, and incon-sistent projections from VM to the S1 incisor representation,although a similar pattern is not noted in other studies in rodents.Thus, despite the presence of an architectonically distinct M1 inrodents, there still appears to be some sensorimotor integrationof the thalamic projections to S1 within most species of rodentsthat have been examined.

SOMATOSENSORY/MOTOR INTEGRATION IN SHORT-TAILED OPOSSUMAs mentioned in the introduction, the short-tailed opossum isunique among mammals investigated in that it does not havea motor representation of its entire body; only the head has acortical motor representation (Frost et al., 2000). These resultsinspired our hypothesis that the body and face representations inS1 would have different patterns of thalamic projections, specif-ically in the case of projections from nuclei associated with themotor system such as VL/VA. As noted above, however, VL/VAonly showed weak and inconsistent projections to S1, and theseweak projections did not differ between S1 face and S1 body(Figures 14C,F). This finding differs from the evidence of a com-plete somatosensory motor amalgam previously described for the






FIGURE 14 | Summary of projections across cortical areas investigated

in these studies. (A) Illustration of cortical areas, using the same colors asthe graphs and diagrams below. Percent of labeled thalamic neurons

(Continued)

FIGURE 14 | Continued

projecting to (B) SR, (C) S1, (D) SC, and (E) MM. Data are mean ± SEMwhen applicable. Axes are the same throughout A-D to aid in comparison.Projections to VPm and VPl are summed above VP, with the projectionsfrom VPm under the horizontal line and the projections from VPl above theline. (F) Summary of strong projections from the 5 cortical areas discussedthroughout the paper. All projections shown make up > 10% of the totalthalamic projections to that area.

Virginia opossum, with extensive projections form VL/VA andVP throughout S1 (Killackey and Ebner, 1973). One explana-tion for these differences between marsupials is that connectionsbetween VL/VA and S1 have been lost in short-tailed opossums;another possibility is that the thalamocortical projection pat-terns in Monodelphis actually represent the ancestral mammalianplan. However, because both thalamic projections and functionalorganization have been assessed in only two opossum species wecannot distinguish between these two alternative hypotheses.

The largest difference we found was that the sensory-motorface representations received moderate projections from VMwhile the S1 body representation did not receive any projectionsfrom VM (Table 3, Figure 14C). In other species, VM receivesinput from the cerebellum and projects to numerous (particularlyrostral) cortical areas as well as other subcortical structures thatare part of the motor system (Donoghue et al., 1979; Herkenham,1979; Kuramoto et al., 2013). In addition to the differential thala-mic projections to the face versus the body representation in S1,we also demonstrate that the face representation receives inputfrom both VPm and VPl while the body representation receivesinput only from VPl (Table 3). In our previous investigation ofcorticocortical connections, we observed that intrinsic connec-tions of S1 are generally heterotropic, and thus not restricted tosimilar somatic representations (Dooley et al., 2013). Thus, theS1 face representation receives projections from thalamic nucleiassociated with motor processing (VM) as well thalamic inputfrom both the face and body somatosensory representations. Thissuggests that motor control of portions of the snout and faceas well as inputs processed within the face representation of S1together form specialized differentiated, highly integrated systemthat differs from the rest of the body.

When considering why the S1 face representation is so highlyintegrated with different cortical areas and thalamic nuclei, it isimportant to consider the peripheral prominence of the vibrissaeand the cortical magnification of the face in light of its ethologicalsignificance. Studies of whisking behavior in short-tailed opos-sums have found that they do display active vibrissal exploratorybehavior (Mitchinson et al., 2011; Grant et al., 2013). This sug-gests that prominent sensory face hairs may have been a sharedfeature of the common ancestor of placental and marsupial mam-mals, and thus may be one of the earliest and most pronouncedforms of sensory reception. Ultimately, the representation of vib-rissae within the cortex may be an example of a significant,and perhaps ubiquitous, site of sensorimotor integration. Asmammalian species radiated, different morphological structuresand associated sensory arrays, such as the hands and eyes inprimates, have undergone specialization and an accompanyingcortical expansion of motor and posterior parietal cortex (see






Cooke et al., 2014). However, the organization and connectivity ofthe opossum vibrissae system suggests that strong sensorimotorintegration of ethologically significant body parts has long been ageneral feature of the mammalian neocortex.

THALAMIC PROJECTIONS TO SR, SC, AND MM IN OTHER SPECIESAmong marsupials, SR and SC have been identified based onreceptive field characteristics and stimulus preference in theVirginia opossum (Beck et al., 1996), the brush-tailed opos-sum (Elston and Manger, 1999), the northern quoll, the stripedpossum (Huffman et al., 1999b), and the big-eared opossum(Anomal et al., 2011). Across the species investigated, neuronsin SR and SC respond predominantly to stimulation of deepreceptors in muscles and joints, show a coarse topographical orga-nization, and have larger receptive fields compared to neurons inS1. In most of these studies, these functionally defined fields havebeen directly related to cortical architecture (see above) and insome species to cortical connections. Corticocortical connectionsof both SR and SC have been reported in the Virginia opossum(Beck et al., 1996), the brush-tailed possum (Elston and Manger,1999), the short-tailed opossum (Dooley et al., 2013), and for SC,the big-eared opossum (Anomal et al., 2011). In all of these stud-ies, the pattern of projections provided further evidence that SRand SC are distinct cortical fields and part of a somatosensorynetwork. It has been proposed that SR is homologous to 3a ordysgranular cortex in rodents and SC is homologous to poste-rior parietal cortex in rats (for review, see Karlen and Krubitzer,2007; Krubitzer et al., 2011). Our studies of corticocortical con-nections as well as previous electrophysiological recording studiesdemonstrate an additional region of cortex just caudal to SC inwhich neurons respond to more than one modality of stimulation(Huffman et al., 1999a; Kahn and Krubitzer, 2002), and whichreceives input from visual and somatosensory cortex (Dooleyet al., 2013). We term this field MM or the multimodal region.

Despite consistent evidence for both SR and SC in all marsupi-als studied, only one study in marsupials examined the thalamo-cortical connections of these fields (Virginia opossum; Donoghueand Ebner, 1981b). In this early study, several injections wereplaced immediately rostral and caudal to S1. The rostral injectionssites were in an area termed post-orbital cortex (likely equiva-lent to SR in the present investigation) and projections to thisarea were from several midline and intralaminar nuclei includingCL, PF, MD, VL, and Pom (identified as CIN), with sparse pro-jections from VP (see Figure 9 in Donoghue and Ebner, 1981b).These projections are consistent with the patterns of projectionswe observed, only with a smaller contribution from CL andmore projections from Pom and VMb. Additionally, while in thepresent investigation only one injection was found to be entirelyrestricted to SR, numerous injections spanned SR and S1 (e.g.,the light green injection in Figure 2A), showing a similar patternof thalamic projections, just with a greater percentage of neuronsoriginating from VP (Figure 14B).

As mentioned above, we have proposed that SR may behomologous to dysgranular cortex (3a) in rodents. In bothrats and squirrels, dysgranular cortex receives strong projec-tions from Pom, along with moderate projections from VP,VL/VA, and CL, as well as projections from VM (Koralek et al.,

1988; Gould et al., 1989). Notably, dysgranular cortex does notreceive projections from midline nuclei MD and PF. However,apart from this exception projections to dysgranular cortex/3aare similar to those to SR in short-tailed and the Virginiaopossums.

Donoghue and Ebner (1981b) did not delineate cortical areascaudal to S1, instead referring to the region caudal to S1 and ros-tral to peristriate cortex (V2) as posterior parietal cortex, or PP.Their injection, however, appears to be in a location similar toMM in the present study rather than to SC, since it borders therostral edge of peristriate cortex (see Figure 8 in Donoghue andEbner, 1981b). They report projections from VL/VA, Pol (termedPo), and weak projections from intralaminar nuclei. Notably, theydo not see any labeled neurons in VP (termed VB) following anyinjections into PP. In the present investigation, both SC and MMreceive strong projections from VL/VA and Pol, as was found inthe Virginia opossum, however projections differ in two impor-tant ways (Figures 14D–F). First, there are still strong projectionsfrom VP to both SC and MM, although not as strong as wasfound for S1. Second, both of these areas (although especiallyMM) display consistent connectivity with visual thalamic nucleiLGNd and the pulvinar. Thus, the general trend of projectionsfrom the thalamus to SC and MM in the short-tailed opossumis consistent with projections described in PP of Virginia opos-sum. However, the short-tailed opossum shows projections fromprimary sensory relay nuclei from both the visual and somatosen-sory systems not found in Virginia opossums, further suggestingthat these thalamic primary sensory nuclei display more exuber-ance in their projections in short-tailed opossums compared toother species.

This exuberance of the projections of these primary sensorynuclei in short-tailed opossums compared to other marsupial andmammalian species builds upon previous work in cortex thatsuggests that brain circuits become more segregated as the brainincreases in size (Ringo, 1991). Increased long range connectionshave been documented in small-brained rodents (Campi et al.,2010; Henschke et al., 2014) as well as primates (Palmer and Rosa,2006). Thus, it is possible that the observed exuberance of tha-lamocortical connectivity in the short-tailed opossum is due tothe small size of their brain. Whether this thalamocortical exu-berance in the small-brained short-tailed opossum is unique tothe species, to small-brained marsupials, or a shared propertyacross other groups of mammals requires a larger comparativeanalysis.

The location and thalamocortical connectivity of MonodelphisMM and SC (PP in Virginia opossums) suggest that theyare homologous to some portion of posterior parietal cortexdescribed in eutherian mammals. Posterior parietal cortex is aregion of the mammalian neocortex which has undergone mas-sive expansion in primates, and thus has been of interest tonumerous researchers interested in the organization of the humanbrain (see Cooke et al., 2014 for review). Despite this, com-paring SC and MM in the present investigation to posteriorparietal areas in other species is challenging, as very little is knownabout the functional role of these areas in opossums and othersmall-brained mammals such as rodents (see Krubitzer et al.,2011). There is, however, an area with similar cortical architecture






and connections that has been identified in rats (termed pos-terior parietal cortex, or PPC; Reep et al., 1994) and squirrels(termed parietal medial area, or Pm; Slutsky et al., 2000). Amongsmall-brained animals, PPC in rats has been the most stud-ied, where, numerous behavioral studies have implicated PPCas part of a larger thalamo-cortical-basal ganglia network thatplays an important role in multimodal spatial attention (Reep andCorwin, 2009). Thalamic projections to PPC include the lateraldorsal nucleus and the lateral posterior nucleus (pulvinar), as wellas projections from VL and Po (Giannetti and Molinari, 2002).Notably, despite close proximity to both S1 and peristriate cortexin rat, PPC does not receive projections from VP or the LGNd.Like PPC in rats, MM in Monodelphis has strong projections fromPol and VL/VA, along with moderate projections with pulvinar(lateral posterior nucleus in rat).

The finding that VL projects to posterior parietal areas has alsobeen described in rats, cats, and monkeys, although it has beensuggested that different populations of neurons within VL areprojecting to motor cortex and parietal areas (Divac et al., 1977;Kasdon and Jacobson, 1978; Hendry et al., 1979; Giannetti andMolinari, 2002). Considering the finding that posterior parietalareas receive projections from VL/VA in the present investiga-tion, we provide additional support for the idea that MM inthe short-tailed opossum may be homologous to portions ofposterior parietal cortex in other mammals, in part due to itsconnectivity to somatosensory, visual and motor nuclei of thethalamus. Further, this finding in Monodelphis provides furtherevidence that the mammalian ancestor of both marsupials andplacentals possessed a cortical area similar to posterior parietalcortex, with a role integrating visual, somatosensory and motorinputs.

CONCLUSIONIn summary, thalamic projections to parietal cortical areas inthe short-tailed opossum are similar not only to other marsu-pials, but other small-brained eutherian mammals. Thus, theseresults provide further evidence for homologies between 3a andSR as well as PP and MM in the short-tailed opossum, sug-gesting that these regions were present in early mammals. Theprojections to these regions, however, differ in short-tailed opos-sums in two notable ways. First, unlike other marsupials studied,there are not extensive projections from VL/VA to S1. This resulthighlights the need for additional comparative studies to moreaccurately infer the cortical organization of the common mam-malian ancestor. Second, in areas such as SR and SC, as well asMM, there are strong projections from VP, along with direct pro-jections from LGNd to MM. This highlights what appears to bea more general feature of the brain of the short-tailed opossum:Increased exuberance of the thalamic projections from primarysensory relay nuclei to non-primary cortical areas. This find-ing suggests that our small-brained mammalian ancestors mayhave also possessed increased thalamocortical connectivity, sim-ilar to the increased cortical connectivity found in many extantsmall-brained mammals. However, we underscore the need foradditional comparative studies of mammals in order to determinewhether this feature has independently evolved in small brainedmammals, or is a retained ancestral trait.

AUTHOR CONTRIBUTIONSAll authors had full access to the data in the study and takeresponsibility for the integrity of the data and the accuracy of thedata analysis. Study concept and design: James C. Dooley, JoãoG. Franca, Dylan F. Cooke, and Leah A. Krubitzer. Acquisition ofdata: James C. Dooley, João G. Franca, Adele M. H. Seelke, DylanF. Cooke, and Leah A. Krubitzer. Histological processing of tissue:James C. Dooley, João G. Franca, Dylan F. Cooke, and Leah A.Krubitzer. Analysis and interpretation of data: James C. Dooley,João G. Franca, Adele M. H. Seelke, Dylan F. Cooke, and Leah A.Krubitzer. Drafting of the article: James C. Dooley and Leah A.Krubitzer. Critical revision of the article for important intellec-tual content: James C. Dooley, João G. Franca, Adele M. H. Seelke,Dylan F. Cooke, and Leah A. Krubitzer. Obtained funding: LeahA. Krubitzer. Study supervision: Leah A. Krubitzer.

FUNDINGThis project was supported by funds to Leah Krubitzer fromNINDS (R21 NS071225) and NEI (R01 EY022987); funds to JoãoFranca from CNPq—Brazil and FAPERJ—Brazil, and funds toJames Dooley from NEI (T32-EY015387-05).

ACKNOWLEDGMENTSThe authors thank Cindy Clayton, UC Davis School forVeterinary Medicine, and the rest of the animal care staff at theUC Davis Psychology Department vivarium. We also thank BeckyGrunewald and for technical assistance; Deepa Ramamurthy andMichaela Donaldson for surgical assistance; Conor Weatherfordfor assistance in XY stage encoding, and Anika K. Colopy foradditional data collection and analysis. Finally, the authors thankMary Baldwin for histological assistance and for helpful com-ments on the manuscript.

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Conflict of Interest Statement: The authors declare that the research was con-ducted in the absence of any commercial or financial relationships that could beconstrued as a potential conflict of interest.

Received: 03 November 2014; accepted: 15 December 2014; published online: 07January 2015.Citation: Dooley JC, Franca JG, Seelke AMH, Cooke DF and Krubitzer LA (2015)Evolution of mammalian sensorimotor cortex: thalamic projections to parietal cor-tical areas in Monodelphis domestica. Front. Neuroanat. 8:163. doi: 10.3389/fnana.2014.00163This article was submitted to the journal Frontiers in Neuroanatomy.Copyright © 2015 Dooley, Franca, Seelke, Cooke and Krubitzer. This is an open-access article distributed under the terms of the Creative Commons Attribution License(CC BY). The use, distribution or reproduction in other forums is permitted, providedthe original author(s) or licensor are credited and that the original publication in thisjournal is cited, in accordance with accepted academic practice. No use, distribution orreproduction is permitted which does not comply with these terms.


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